Glynn Wilson

The galactose-specific recognition system of mammalian liver: The route of ligand internalization in rat hepatocytes

We have used two electron microscopic tracers, asialoorosomucoid covalently coupled to horseradish peroxidase (ASOR-HRP) and lactosaminated ferritin (Lac-Fer), to investigate the internalization of proteins bound by the asialoprotein receptor of rat hepatocytes. Both ligands are cleared rapidly from the circulation of rats, are retarded in their clearance by an excess of ASOR and accumulate principally in the liver. Morphological examination of the livers of rats after injection of the probes confirmed that the hepatocyte is the principal liver cell involved in the clearance of galactose-terminating proteins. Internalization occurred via coated pits and coated vesicles of 1000 A diameter. At 30 sec to 2 min the tracers began to accumulate in a complex arrangement of larger smooth-surfaced vesicles and tubular structures at the sinusoidal periphery of the cell. Fluid phase pinocytosis did not appear to account for any of the uptake into larger vesicles. The particulate tracer, Lac-Fer, was closely apposed to the membrane of coated pits and vesicles, but was found scattered throughout the lumen of the larger vesicles, possibly indicating dissociation of the ligand from its receptor. Although occasional lysosomes were detected cytochemically in the cell periphery, vesicles containing Lac-Fer showed no demonstrable aryl sulfatase activity. At 5 min, the tracers began to appear in Golgi-lysosome regions of the hepatocyte and were present in small vesicles of <2000 A in diameter, larger irregular vesicles and tubules. Serial sectioning indicated that tubular structures in Golgi-lysosome regions were often interconnected to the larger vesicles, but that tubules in the peripheral cytoplasm were only occasionally connected to larger structures. Some of the Lar-Fer-containing vesicles in Golgi-lysosome areas at 15 min after injection were found to contain aryl sulfatase reaction product, indicating fusion with lysosomes.

Models for assessing drug absorption and metabolism

General Principles in the Characterization and Use of Model Systems for Biopharmaceutical Studies R.T. Borchardt, et al. Methods for Evaluating Intestinal Permeability and Metabolism in vitro P.L. Smith. Culturedintestinal Epithelial Cell Models I.J. Hidalgo. Intestinal Rings and Isolated Intestinal Mucosal Cells J.A. Fix. Models of Drug Absorption in situand in Conscious Animals R. Griffiths, et al. Model Systems for Intestinal Lymphatic Transport Studies C.J.H. Porter, W.N. Charman. Buccal Tissues and Cell Culture E. Quadros, et al. Isolated Hepatocytes M. Vore, et al. Cultured Rat Hepatocytes E.L. Le Cluyse, et al. Isolated Perfused Liver K.L.R. Brouwer,R.G. Thurman. Isolated Renal Brush Border and Basolateral Membrane Vesicles and Cultured Renal Cells M.M. Gutierrez, et al. Use of an Isolated Perfused Kidney to Assess Renal Clearance of Drugs: Information Obtained in Steadystate and Nonsteady State Experimental Systems N. Okudaira, Y. Sugiyama. Brain Microvessel Endothelial Cell Culture Systems K.L. Audus, et al. Methods to Study Drug Transport in Isolated Choroid Plexus Tissue and Cultured Cells C.B. Washington, et al. Brain Perfusion Systems for Studies of Drug Uptake and Metabolism in the Central Nervous System Q.R. Smith. 8 additional articles. Index.

In Vitro Nasal Transport Across Ovine Mucosa: Effects of Ammonium Glycyrrhizinate on Electrical Properties and Permeability of Growth Hormone Releasing Peptide, Mannitol, and Lucifer Yellow

Transport of growth hormone releasing peptide across ovine nasal mucosa in the absence or presence of ammonium glycyrrhizinate (AMGZ) was studied in vitro. Ovine nasal mucosa was stripped from underlying cartilage and mounted in Ussing chambers. Transepithelial conductance (Gt) and short-circuit current (Isc) were monitored during experiments to assess tissue viability and integrity. Radiolabeled mannitol (Man; MW 182) and growth hormone releasing peptide (GHRP, SK&F 110679; MW 873) were employed to measure transport rates across the epithelium, and fluorescence spectroscopy was employed to measure rates of lucifer yellow (LY; MW 521) transport. Effects of AMGZ on ovine nasal mucosal viability and transport were determined from changes in electrical properties or fluxes of [3H]GHRP, [3H]Man, and LY. Results demonstrate that electrical properties of ovine nasal mucosa are stable over the time course of the experiments (Gt = 8.3 ± 0.5 mS/cm2 and Isc = 3.7 ± 0.2 µEq/hr · cm2; n = 21). Man fluxes were comparable in the mucosal (m)-to-serosal (s) and s-to-m directions [0.10 ± 0.01 (n = 17) and 0.10 ± 0.01 (n = 4) %/hr · cm2, respectively]. Transport of GHRP and LY in the m-s direction was similar to that of Man [0.08 ± 0.01 (n = 11) and 0.09 ± 0.01 (n = 3) %/hr · cm2, respectively]. GHRP flux was equivalent in the m-s and s-m directions. GHRP did not significantly alter ion transport processes as indicated by the lack of any change in Gt or Isc. Luminal addition of AMGZ (2%, 24 mM) increases Gt and transport of both LY and [3H]Man approximately fourfold without altering transport of [3H]GHRP. No changes in transport or Gt were seen with luminal addition of AMGZ (1%, 12 mM). These studies suggest that transport of the hexapeptide GHRP occurs by a passive process and that AMGZ selectively increases the permeability of the mucosa to the low molecular weight molecules, Man and LY, but not to GHRP in vitro.

Chemical Approaches to Improve the Oral Bioavailability of Peptidergic Molecules

This review discusses both tools and strategies that may be employed as approaches towards the pursuit of orally active compounds from peptidergic molecules. Besides providing a review of these subjects, this paper provides an example of how these were utilized in a research programme at SmithKline Beecham involving the development of orally active GPIIb/IIIa antagonists. The tools for studying oral drug absorption in‐vitro include variants of the Ussing chamber which utilize either intestinal tissues or cultured epithelial cells that permit the measurement of intestinal permeability. Example absorption studies that are described are mannitol, cephalexin, the growth hormone‐releasing peptide SK&F 110679 and two GPIIb/ IIIa antagonist peptides SK&F 106760 and SK&F 107260. With the exception of cephalexin, these compounds cross the intestine by passive paracellular diffusion. Cephalexin, on the other hand, crosses the intestine via the oligopeptide transporter. Structure‐transport studies are reviewed for this transporter. The tools for studying oral drug absorption in‐vivo involve animals bearing in‐dwelling intestinal or portal vein catheters. A study of the segmental absorption of SK&F 106760 is provided.

Exploitation of the Intestinal Oligopeptide Transporter to Enhance Drug Absorption

AbstractStudies of the mechanisms involved in the transport of di- and tri-peptides by the intestinal oligopeptide transporter suggest a process which involves proton-dependent uptake at the apical cell membrane of the enterocytes with subsequent exit of intact di- or tri-peptides across the basolateral membrane or, alternatively, intracellular hydrolysis and exit of component amino acids across the basolateral membrane. In the development of techniques for investigating interaction of molecules with this transporter, it was demonstrated that peptidomimetics such as β-lactam antibiotics, cephalosporins, angiotensin converting enzyme inhibitors, and renin inhibitors are taken up at the apical cell membrane of entrocytes by the oliopeptide transporter. Molecules which interact with and are taken up by the oligopeptide transporter demonstrate good oral absorption. In addition, prodrugs of α-methyldopa and phosphonoformic acid which interact with the intestinal oligopeptide transporter and which have enhanced...

A comparison of the affinities of dipeptides and antibiotics for the di-/tripeptide transporter in Caco-2 cells

Abstract The intestinal peptide transporter(s) is involved in the absorption of natural di-/tripeptides and peptidomimetic drugs. Several key aspects of peptide transport such as number of peptide transporters and structural requirements for transport via this carrier(s) are not fully understood. In addition, recent studies showed that interaction with the di-/tripeptide transporter(s) does not necessarily lead to transcellular transport. The variety of structures which appear to interact with the transporter could be explained by the presence of several transporters or by multiple binding sites in a single transporter. The objective of this study was to determine whether there is a clear difference between dipeptides and amino-β-lactam antibiotics which may suggest the involvement of different transporters/binding sites. Experiments were carried out in Caco-2 cell monolayers grown on microporous membranes using cephalexin as the model compound. Inhibition constants ( K i ) were calculated from Dixon plots assuming competitive inhibition. The strong correlation between K i and IC 50 (independently determined) indicates that the assumption of competitive inhibition was probably correct. Results show: (a) that dipeptides have greater affinity for the cephalexin transporter(s) than antibiotics; (b) among dipeptides, neutral dipeptides seem to have higher affinity for the carrier; and (c) a relationship between affinity for the transporter(s) and transepithelial transport could not be found.

An in vitro pulmonary epithelial system for evaluating peptide transport

Abstract A number of in vitro cell and tissue culture methodologies have been employed to study the potential exploitation of transport mechanisms for the delivery of peptidergic molecules across the oral and respiratory epithelia. In contrast to the development of in vitro methodologies to study transport across the intestinal and nasal mucosae, a convenient and well characterized system to study peptide transport across the pulmonary epithelium remains to be achieved. Further impetus for developing such a system stems from recent observations of high systemic bioavailability of peptidergic molecules after pulmonary administration, and the need for further studies on potential routes and mechanisms of transport in the lung. We have investigated the utility of Xenopus lung mounted in Ussing chambers for such studies. Xenopus lung resembles mammalian lung morphologically and physiologically, including: (1) similar composition and dimensions of the air-blood barrier, (2) high transepithelial resistance and (3) surfactant production by pulmonary epithelial cells. Tissue viability can be maintained over 4 h in vitro as assessed by constant I sc , constant average resistance, decrease of I sc in response to amiloride or ouabain, and normal ultrastructural morphology. Transport of several model hydrophilic and hydrophobic compounds was linear over 3 h. Relative rates of mannitol, inulin, leucine, and antipyrine transport correlated well with their relative rates of disappearance from rat lungs in vivo. Thus the properties of this system make it appropriate for use in analyzing the rates and mechanisms of peptide transport across the pulmonary epithelium.

General Principles in the Characterization and Use of Model Systems for Biopharmaceutical Studies

A major challenge confronting pharmaceutical chemists in the future will be the design of drug candidates having structural characteristics adequate to circumvent the biological barriers [e. g., intestinal mucosa, liver, blood-brain barrier (BBB)] that often prevent the clinical development of potentially useful drug candidates (Audus and Raub, 1993). Through rational drug design, medicinal chemists are capable of synthesizing very potent and very specific drug candidates (Morgan and Gainer, 1989; Greenlee, 1990; Huff, 1991; Doherty, 1992; Bondinell et al., 1994). These drug candidates are developed with molecular characteristics that permit optimal interaction with the specific macromolecules (e. g., receptors, enzymes) that mediate their pharmacological effects. However, rational drug design, as currently practiced in many pharmaceutical companies, does not necessarily ensure optimal delivery of the drug to its site of action. Optimal delivery can be achieved by incorporating the drug candidate into a delivery system (e. g., formulation strategies) and/or by designing the drug candidate to have the structural characteristics (rational drug design strategies) that will provide optimal transfer between the point of administration and the pharmacological target in the body (Lee, 1991).